2.1 Leukocyte Extravasation
The migration of leukocytes (white blood cells) out of the blood and into tissues (extravasation) is the central event in the inflammatory response. Leukocyte emigration is responsible for the successful host response to tissue injury and infection, but is also potentially harmful and contributes to the pathology of many diseases and inflammatory disorders.
Lymphocytes exit from the blood by selective interaction with high endothelial venule (HEV) cells. In autoimmune disease and inflammation, most lymphocyte extravasation occurs through nonspecialized endothelium rather than HEV. Recirculating lymphocytes express homing receptors which interact in an organ-specific manner with HEV in peripheral lymph nodes, mucosa-associated lymphoid tissues, and in inflamed joint tissue (Jalkanen et al., 1987, Ann. Rev. Med. 38: 467-476). Related receptors are expressed on other leukocyte subsets (id.). The first step in leukocyte migration into tissues is margination, when leukocytes leave the central stream of flowing blood cells in a postcapillary venule and roll along the endothelial lining of the vessel (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)). Leukocytic margination in postcapillary venules should be distinguished from the "marginating pool" of about 50% of leukocytes that may be in capillary beds in the lung or tissues and enter the circulation in response to exercise or epinephrine. Postcapillary venules are major sites of leukocyte emigration in inflammation, and there are few or no marginating leukocytes in these venules in the healthy state (Fiebig et al., 1991, Int. J. Microcirc. Clin. Exp. 10: 127-144).
As observed more than 100 years ago using intravital microscopy (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)), leukocytes begin to interact with the vessel wall by rolling along the endothelium within minutes after injury to adjacent tissue. The rolling response is seen throughout Vertebrata, in cold-blooded animals such as amphibians as well as in mammals (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)). The number of rolling cells increases dramatically during the course of an inflammatory reaction (Atherton and Born, 1972, J. Physiol. 233: 157-165) and is important in the accumulation of cells at the site (Fiebig et al., 1991, Int. J. Microcirc. Clin. Exp. 10: 127-144). As the inflammatory reaction progresses, the endothelium becomes paved with leukocytes, and their rolling decreases in velocity and is interrupted by halts until they come to a firm stop (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)). Throughout this process the cells remain round, but undergo a dramatic change in shape immediately upon initiation of emigration. A pseudopod is extended through the vessel at a junction between endothelial cells, and this often is accompanied by a flattening of the leukocyte against the vessel wall (Marchesi, 1961, Q. J. Exp. Physiol. 46: 115-133). Transmigration continues as the pseudopod grows in ramifications and size until the entire cell body has emerged through a narrow gap between endothelial cells (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)). Cells appear to reach the point at which they emigrate by rolling; no active migration along the vessel wall is evident by intravital microscopy.
Both the rheology of blood and specific adhesive interactions may regulate the rolling response. Hydrodynamic studies of particles in suspension show that in Poiseuille flow, the larger particles are forced to the center of the stream, and this effect is more pronounced as shear forces increase (Segre and Silberberg, 1962, J. Fluid Mech. 14: 136-157). This effect has been confirmed for blood cells both in vivo and in vitro; the larger leukocytes are forced to the center of the stream in normal flow (Goldsmith and Spain, 1984, Microvasc. Res. 27: 204-222; Nobis et al., 1985, Microvasc. Res. 29: 295-304). In inflammation, vessels dilate and flow is slowed. Vascular permeability is increased, leading to plasma leakage and an increased hematocrit, and together with slower flow, leads to erythrocyte rouleaux formation. A combination of these factors causes leukocytes to be displaced to the marginal region of flow near the vessel wall (Chien, 1982, Adv. Shock Res. 8: 71-80). This makes contact of a circulating leukocyte with the vessel wall more probable, but shear forces acting on the leukocyte at the vessel wall oppose adhesion to the endothelium. The velocity profile of a vessel shows no flow at the vessel wall and a parabolic increase toward the centerline. Because fluid velocity increases with distance from the wall, cells near the wall have torque exerted on them and will tumble even if not in contact with the wall. However, the velocity at which cells tumble in a shear flow near to the vessel wall is much faster than observed for rolling cells in inflammatory reactions, suggesting that adhesive interactions occur between the leukocyte and vessel endothelium (Atherton and Born, 1973, J. Physiol. 233: 157-165).
More than 100 years after Cohnheim (1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)) postulated molecular changes in vessel endothelium in inflammation, the molecular basis of leukocyte rolling remains unknown. However, three families of adhesion receptors that participate in leukocyte interactions with endothelium have been defined: the integrin, immunoglobulin-related, and selectin molecules (reviewed in Springer, 1990, Nature 346: 425-433)).
The integrins LFA (lymphocyte function-associated antigen)-1 and Mac-1 on the neutrophil bind to the Ig family member ICAM (intercellular adhesion molecule)-1 on endothelium (Smith et al., 1988, J. Clin. Invest. 82: 1746-1756; Smith et al., 1989, J. Clin. Invest. 83: 2008-2017; Diamond et al., 1990, J. Cell Biol. 111: 3129-3139). LFA-1 and not Mac-1 binds to ICAM-2 (de Fougerolles et al., 1991 J. Exp. Med. 174: 253-267; Diamond et al., 1990, J. Cell Biol. 111: 3129-3139), an endothelial cell molecule that is more closely related to ICAM-1 than these molecules are to other Ig superfamily members (Staunton et al., 1989, Nature 339: 61-64). Stimulation of neutrophils with chemoattractants is required to activate binding of these integrins to ICAM-1 (Smith et al., 1989, J. Clin. Invest. 83: 2008-2017; Diamond et al., 1990, J. Cell Biol. 111: 3129-3139). Stimulation of neutrophil integrin avidity is a rapid response occurring in minutes, does not require increased integrin surface expression (Buyon et al., 1988, J. Immunol. 140: 3156-3160; Philips et al., 1988, J. Clin. Invest. 82: 495-501; Vedder and Harlan, 1988, J. Clin. Invest. 81: 676-682; Lo et al., 1989, J. Exp. Med. 169: 1779-1793), and appears analogous to an increase in avidity described for LFA-1 on T lymphocytes in response to antigen receptor crosslinking (Dustin and Springer, 1989, J. Cell Biol. 107: 321-331).
ICAM-1 induction is a second mechanism for regulating inflammatory cell interactions that occurs on a time scale of hours and requires mRNA and protein synthesis (reviewed in Springer, 1990, Nature 346: 425-433). ICAM-1 is expressed basally on endothelial cells but is greatly increased at inflammatory sites and by stimulation with lipopolysaccharide and cytokines such as IL-1 and TNF. By contrast to ICAM-1, ICAM-2 is expressed at higher surface density on resting endothelium but is not inducible (de Fougerolles et al., 1991, J. Exp. Med. 174: 253-267).
LFA-1 and Mac-1 together with p150,95 comprise the leukocyte integrins, a subfamily of integrins that share a common .beta. subunit (CD18) and have distinct .alpha.L, .alpha.M and .alpha.X (CD11a, b and c) .alpha. subunits (reviewed in Larson and Springer, 1990, Immunol. Rev. 114: 181-217; Springer, 1990, Nature 346: 425-433). They are required for leukocyte emigration as demonstrated by an absence of neutrophil extravasation 1) in patients with mutations in the common .beta. subunit (leukocyte adhesion deficiency), and 2) after treatment of healthy neutrophils with a monoclonal antibody (mAb) to the common .beta. subunit in vivo or in vitro (reviewed in (Anderson and Springer, 1987, Ann. Rev. Med. 38: 175-194; Larson and Springer, 1990, Immunol. Rev. 114: 181-217). Patient neutrophils, and healthy neutrophils treated with mAb to the common .beta. subunit or a combination of mAb to LFA-1 and Mac-1 .alpha. subunits, are deficient in binding to endothelial cells in static adhesion assays (Buchanan et al., 1982, Blood 60: 160-165; Harlan et al., 1985, Blood 66: 167-178). However, when binding of neutrophils in shear flow is measured, the leukocyte integrin-dependent component of binding is lost at a shear stress below the physiologic range (Lawrence et al., 1990, Blood 75: 227-237). Nonetheless, patient and CD18-treated cells that bind to the endothelium through other adhesion mechanisms fail to undergo transendothelial migration, in agreement with the lack of neutrophil diapedesis in leukocyte adhesion deficiency (Smith et al., 1988, J. Clin. Invest. 82: 1746-1756).
The integrin VLA-4, that contains the .alpha.4 (CD49d) subunit noncovalently associated with the .beta.1 (CD29) subunit, is expressed by lymphocytes, monocytes, and neural crest-derived cells, and can interact with vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., 1990, Cell 60: 577). Like ICAM-1 and ICAM-2, VCAM-1 is a member of the immunoglobulin (Ig) superfamily (Osborn et al., 1989, Cell 59: 1203), but unlike the ICAMs, VCAM-1 is not expressed by lymphocytes (Wellicome et al., 1990, J. Immunol. 144: 2558; Rice et al., 1990, J. Exp. Med. 171: 1369). VCAM-1 expression is very low or absent on resting endothelial cells in culture but can be induced by cytokines such as TNF or IL-1 with kinetics of induction similar but not identical to that of ICAM-1 (Wellicome et al., 1990, J. Immunol. 144: 2558; Carlos et al., 1990, Blood 76: 965). Peak expression of VCAM-1 after continuous treatment of endothelial cells with TNF in culture occurs somewhat earlier than the peak expression of ICAM-1, but both persist at levels substantially higher than basal expression for at least 48 hr (Carlos et al., 1990, Blood 76: 965). Unlike LFA-1, however, VLA-4 can also interact with fibronectin, binding to the alternatively spliced CS-1 region located C-terminal to the RGD site of fibronectin recognized by the integrin VLA-5 (Guan and Hynes, 1990, Cell 60: 53; Wayner et al., 1989, J. Cell Biol. 109: 1321; Hemler, 1990, Annu. Rev. Immunol. 8: 365). Two forms of VCAM-1 cDNA clones, which most likely represent alternatively spliced products, have been reported (Osborn et al., 1989, Cell 59: 1203; Polte et al., 1990, Nucl. Acids Res. 18: 5901; Cybulsky et al., 1991, Am. J. Pathol. 138: 815; Hession et al., 1991, J. Biol. Chem. 266: 6682).
The selectins are the most recently recognized class of leukocyte adhesion molecules (reviewed in Springer, 1990, Nature 346: 425-433). They have an N-terminal lectin domain, one epidermal growth factor-like module, and from two to nine short consensus repeats. By contrast to integrins and immunoglobulin (Ig) family members, selectins have been found to date only on circulating cells and the endothelium, suggesting that they may be specialized for interactions within the vasculature. CD62 (PADGEM or GMP-140) is expressed in .alpha. granules of platelets and Weibel-Palade bodies of endothelial cells, and is mobilized to the plasma membranes of these cells after activation by mediators of inflammation and hemostasis, allowing these cells to bind neutrophils and monocytes at the site of tissue injury (Larsen et al., 1989, Cell 59: 305-312; Geng et al., 1990, Nature 343: 757-760). CD62 is rapidly unregulated on the endothelial cell surface, suggesting that it may be important early in inflammation (Hattori et al., 1989, J. Biol. Chem. 264: 7768-7771; Geng et al., 1990, Nature 343: 757- 760). ELAM-1 is synthesized by endothelial cells in response to inflammatory agents and promotes adhesion of neutrophils, monocytes, and a subpopulation of lymphocytes (Bevilacqua et al., 1989, Science 243: 1160-1165; Picker et al., 1991, Nature 349: 796-798; Shimizu et al., 1991, Nature 349: 799-802). The LAM-1 or LECAM-1 molecule is expressed on leukocytes and facilitates their binding to endothelium during lymphocyte recirculation through peripheral lymph nodes and neutrophil emigration at inflammatory sites (Jutila et al., 1989, J. Immunol. 143: 3318-3324; Spertini et al., 1991, Nature 349:691-694; Watson et al., 1991, Nature 349: 164-167). Carbohydrate ligands for selectins have recently been defined (reviewed in Springer and Lasky, 1991, Nature 349: 196-197); that for CD62 has Lewis x as an important component (Larsen et al., 1990, Cell 63: 467-474) and also appears to be sialylated (Moore et al., 1991, J. Cell Biol. 112: 491-499). Neutrophils bear Lewis x both on glycolipids and at the termini of N- and O-linked oligosaccharides (Symington et al., 1985, J. Immunol. 134: 2498-2506; Fukuda et al., 1984, J. Biol. Chem. 259: 10925-10935). Antibodies to selectins and integrins additively inhibit neutrophil adhesion to endothelium, suggesting that they mediate distinct adhesion mechanisms (Luscinskas et al., 1989, J. Immunol. 142: 2257-2263; Dobrina et al., 1989, Immunology 67: 502-508; Smith et al., 1991, J. Clin. Invest. 87: 608-618; Hallmann et al., 1991, Biochem. Biophys. Res. Commun. 174: 236-243). The molecular basis of rolling does not appear to involve the leukocyte integrins, based on the inability of Mab to the leukocyte integrin common CD 18 .beta. subunit to inhibit rolling in vivo (Arfors et al., 1987, Blood 69: 338-340).
Chemoattractants bind to serpentine family receptors on the surface of a leukocyte. A highly selective class of chemoattractants described in the last few years are small proteins of 70 to 80 amino acids that belong to a recently described protein family called the intercrines (Oppenheim et al., 1991, Ann. Rev. Immunol. 9: 617-648). The chemoattractant receptors mediate pro-inflammatory and chemotactic actions, and transduce ligand-mediated signals through interactions with G proteins (GTP-binding proteins). Actions mediated by chemoattractant receptors include stimulation of granule-enzyme release and superoxide anion production, upregulation of expression and activity of the cell adhesion molecule Mac-1 (CDIIb, CD18), increased expression of CR1, a decrease in cell surface glycoprotein 100MEL-14 on neutrophils (Gerard and Gerard, 1991, Nature 349: 6-14), and stimulation of neutrophil adherence to and emigration through activated endothelial cells (Huber et al., 1991, Science 254: 99). Interleukin (IL-8) can also act as an adhesion or migration inhibitor when added on the same side of activated endothelium as neutrophils (Huber et al., 1991, Science 254: 99; Gimbrone et al., 1989, Science 246: 1601). In vivo, these receptors may participate in anaphylactoid and septic shock (Gerard and Gerard, supra).
The best characterized chemoattractant receptor is the one which binds formylpeptides. cDNAs encoding receptors for four chemoattractants, formylpeptide [e.g. fMet-Leu-Phe (fMLP)] (Boulay et al., 1990, Biochem. Biophys. Res. Commun. 168:1103-1109; Boulay et al., 1990, Biochemistry 29:11123-11133), C5a (Gerard and Gerard, 1991, Nature 349: 614-617) platelet activating factor (PAF; Honda et al., 1991, Nature 349: 342-346), and IL-8 (Holmes et al., 1991, Science 253: 1278-1280) have been cloned.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.